木瓜发酵物通过miR-350-3p/TLR4信号通路改善小鼠非酒精性脂肪性肝炎

Chaenomeles speciosa Fermentation Improves Nonalcoholic Steatohepatitis in Mice via miR-350-3p/TLR4 Pathway

目的研究木瓜发酵物(CSF)对高脂饲料(HFD)联合葡聚糖硫酸钠(DSS)诱导的小鼠非酒精性脂肪性肝炎(NASH)的改善作用,探讨木瓜发酵物对非酒精性脂肪性肝炎小鼠miR-350-3p/Toll样受体4(TLR4)信号通路的调控机制。方法采用益生菌发酵皱皮木瓜,应用高脂饲料联合葡聚糖硫酸钠诱导小鼠非酒精性脂肪性肝炎模型。将SPF级雄性昆明小鼠随机分为正常组、模型组及木瓜发酵物低、高剂量组,每组10只。灌胃给药,每天1次,连续20 d。其间,正常组给予正常饲料,模型组和木瓜发酵物组均给予高脂饲料,模型组及木瓜发酵物组在最后5 d连续给予2.5%葡聚糖硫酸钠。计算小鼠肝指数;检测小鼠血清甘油三酯(TG)、胆固醇(CHO)含量;HE染色法观察小鼠肝脏组织病理学变化;采用RT-qPCR法检测小鼠肝脏组织中miR-350-3p、TLR4、白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)表达情况;Western Blot法分析小鼠肝脏中TLR4、TNF-α蛋白表达情况。结果与正常组比较,模型组小鼠肝指数、甘油三酯和胆固醇均有升高(P<0.05),HE染色结果显示肝脏出现明显脂肪变性和炎症反应,RT-qPCR结果显示TLR4和炎性因子(IL-6、TNF-α)表达明显升高(P<0.05),而miR-350-3p表达降低(P<0.05);与模型组比较,木瓜发酵物可使小鼠肝指数、肝脏甘油三酯和胆固醇明显降低(P<0.05),TLR4和炎性因子(IL-6、TNF-α)表达降低(P<0.05),而miR-350-3p表达升高(P<0.05)。结论木瓜发酵物可有效改善高脂饲料联合葡聚糖硫酸钠诱导的小鼠非酒精性脂肪肝炎的炎症反应,其机制可能与调控miR-350-3p/TLR4信号通路有关。

Objective To investigate the effects of Chaenomeles speciosa fermentation(CSF) on nonalcoholic steatohepatitis(NASH) induced by high-fat diet(HFD) and dextran sodium sulfate(DSS), and to explore the mechanism of CSF in regulating miR-350-3 p/TLR4 signaling pathway in NASH mice. Methods In this study,the NASH model was induced by HFD combined with DSS in mice, and then treated with Chaenomeles speciosa fermented by probiotics yeast. SPF male Kunming mice were randomly divided into 4 groups with 10 mice in each group,including control group,model group,and CSF low-,high-dose groups. Intragastric administration was performed once a day for 20 d,The control group was given normal diet,the model group and CSF groups were simultaneously given HFD,and 2.5% DSS was administered continuously for the last 5 days in the model and CSF groups. The liver index and the contents of triglyceride(TG)and cholesterol(CHO)in serum were detected in mice.Histopathological changes of liver were observed by HE staining. The expression of miR-350-3 p, Toll-like receptor 4(TLR4),IL-6 and TNF-α in mouse liver tissues was detected by RT-qPCR. The protein expression of TLR4 and TNF-α in liver of mice was analyzed by Western Blot. Results Compared with the normal group,the liver index, triglyceride(TG) and cholesterol(CHO) in the model group were increased(P<0.05). HE staining showed significant liver steatosis and inflammation in model mice. The expression of Toll-like receptor 4(TLR4)and inflammatory factors(IL-6,TNF-α)was significantly increased,while miR-350-3 p expression was decreased in the liver tissues(P<0.05). Compared with the model group,CSF significantly decreased liver index,TG and CHO(P<0.05),the expression of TLR4,IL-6 and TNF-α,and increased the expression of miR-350-3 p(P<0.05).Conclusion CSF can effectively improve the inflammatory response of NASH induced by HFD combined with DSS.The mechanism may be related to the regulation of miR-350-3 p/TLR4 signaling pathway.